Genes that regulate hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation are tightly controlled by regulatory regions. However, mapping such regions relies on surface markers and immunophenotypic definition of HSCs. Here, we use γ-retroviral integration sites (γRV ISs) from a gene therapy trial for 10 patients with Wiskott-Aldrich syndrome to mark active enhancers and promoters in functionally defined long-term repopulating HSCs. Integration site clusters showed the highest ATAC-seq signals at HSC-specific peaks and strongly correlated with hematopoietic risk variants. Tagged genes were significantly enriched for HSC gene sets. We were able to map over 3,000 HSC regulatory regions in late-contributing HSCs, and we used these data to identify miR-10a and miR-335 as two miRNAs regulating early hematopoiesis. In this study, we show that viral insertion sites can be used as molecular tags to assess chromatin conformation on functionally defined cell populations, thereby providing a genome-wide resource for regulatory regions in human repopulating long-term HSCs.

Elucidating how cannabinoids affect brain function is instrumental for the development of therapeutic tools aiming to mitigate 'on target' side effects of cannabinoid based therapies. A single treatment with the cannabinoid receptor agonist, WIN 55,212-2, disrupts recognition memory in mice. Here we evaluate how prolonged, intermittent (30 days) exposure to WIN 55,212-2 (1mg/kg) alters recognition memory and impacts on brain metabolism and functional connectivity. We show that chronic, intermittent treatment with WIN 55,212-2 disrupts recognition memory (Novel Object Recognition Test) without affecting locomotion and anxiety-like behaviour (Open Field and Elevated Plus Maze). Through 14 C-2-deoxyglucose functional brain imaging we show that chronic, intermittent WIN 55,212-2 exposure induces hypometabolism in the hippocampal dorsal subiculum and in the mediodorsal nucleus of the thalamus, two brain regions directly involved in recognition memory. In addition, WIN 55,212-2 exposure induces hypometabolism in the habenula with a contrasting hypermetabolism in the globus pallidus. Through the application of the Partial Least Squares Regression (PLSR) algorithm to the brain imaging data, we observed that prolonged WIN 55,212-2 administration alters functional connectivity in brain networks that underlie recognition memory, including that between the hippocampus and prefrontal cortex, the thalamus and prefrontal cortex, and between the hippocampus and the perirhinal cortex. In addition, our results support disturbed lateral habenula and serotonin system functional connectivity following WIN 55,212-2 exposure. Overall, this study provides new insight into the functional mechanisms underlying the impact of chronic cannabinoid exposure on memory and highlights the serotonin system as a particularly vulnerable target. This article is protected by copyright. All rights reserved.

Activity-dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery to maintain protein homeostasis, critical for proper neuronal function. However, the biochemical evidence for this balance and coordination is largely lacking. Leveraging our recent discovery of a neuronal-specific 20S membrane proteasome complex (NMP), we began exploring how neuronal activity regulates its function. Here, we found that the NMP degrades exclusively a large fraction of ribosome-associated nascent polypeptides that are being newly synthesized during neuronal stimulation. Using deep-coverage and global mass spectrometry, we identified the nascent protein substrates of the NMP, which included products encoding immediate-early genes, such as c-Fos and Npas4. Intriguingly, we found that turnover of nascent polypeptides and not full-length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity-induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP.

Salt intake is an essential dietary requirement, but excessive consumption is implicated in hypertension and associated conditions. Little is known about the neural circuit mechanisms that control motivation to consume salt, although the midbrain dopamine system, which plays a key role in other reward-related behaviors, has been implicated. We, therefore, examined the effects on salt consumption of either optogenetic excitation or chemogenetic inhibition of ventral tegmental area (VTA) dopamine neurons in male mice. Strikingly, optogenetic excitation of dopamine neurons decreased salt intake in a rapid and reversible manner, despite a strong salt appetite. Importantly, optogenetic excitation was not aversive, did not induce hyperactivity, and did not alter salt concentration preferences in a need-free state. In addition, we found that chemogenetic inhibition of dopamine neurons had no effect on salt intake. Lastly, optogenetic excitation of dopamine neurons reduced consumption of sucrose following an overnight fast, suggesting a more general role of VTA dopamine neuron excitation in organizing motivated behaviors.

A vast number of different neuronal activity patterns could each induce a different set of activity-regulated genes. Mapping this coupling between activity pattern and gene induction would allow inference of a neuron's activity-pattern history from its gene expression and improve our understanding of activity-pattern-dependent synaptic plasticity. In genome-scale experiments comparing brief and sustained activity patterns, we reveal that activity-duration history can be inferred from gene expression profiles. Brief activity selectively induces a small subset of the activity-regulated gene program that corresponds to the first of three temporal waves of genes induced by sustained activity. Induction of these first-wave genes is mechanistically distinct from that of the later waves because it requires MAPK/ERK signaling but does not require de novo translation. Thus, the same mechanisms that establish the multi-wave temporal structure of gene induction also enable different gene sets to be induced by different activity durations.

Traumatic axonal injury (TAI) is a common neuropathology in traumatic brain injury and is featured by primary injury to axons. Here, we generated TAI with impact acceleration of the head in male Thy1-eYFP-H transgenic mice in which specific populations of neurons and their axons are labeled with yellow fluorescent protein. This model results in axonal lesions in multiple axonal tracts along with blood-brain barrier disruption and neuroinflammation. The corticospinal tract, a prototypical long tract, is severely affected and is the focus of this study. Using optimized CLARITY at single-axon resolution, we visualized the entire corticospinal tract volume from the pons to the cervical spinal cord in 3D and counted the total number of axonal lesions and their progression over time. Our results divulged the presence of progressive traumatic axonopathy that was maximal at the pyramidal decussation. The perikarya of injured corticospinal neurons atrophied, but there was no evidence of neuronal cell death. We also used CLARITY at single-axon resolution to explore the role of the NMNAT2-SARM1 axonal self-destruction pathway in traumatic axonopathy. When we interfered with this pathway by genetically ablating SARM1 or by pharmacological strategies designed to increase levels of Nicotinamide (Nam), a feedback inhibitor of SARM1, we found a significant reduction in the number of axonal lesions early after injury. Our findings show that high-resolution neuroanatomical strategies reveal important features of TAI with biological implications, especially the progressive axonopathic nature of TAI and the role of the NMNAT2-SARM1 pathway in the early stages of axonopathy.SIGNIFICANCE STATEMENT In the first systematic application of novel high-resolution neuroanatomical tools in neuropathology, we combined CLARITY with 2-photon microscopy, optimized for detection of single axonal lesions, to reconstruct the injured mouse brainstem in a model of traumatic axonal injury (TAI) that is a common pathology associated with traumatic brain injury. The 3D reconstruction of the corticospinal tract at single-axon resolution allowed for a more advanced level of qualitative and quantitative understanding of TAI. Using this model, we showed that TAI is an axonopathy with a prominent role of the NMNAT2-SARM1 molecular pathway, that is also implicated in peripheral neuropathy. Our results indicate that high-resolution anatomical models of TAI afford a level of detail and sensitivity that is ideal for testing novel molecular and biomechanical hypotheses.

Plasmodium infection begins with the bite of an anopheline mosquito, when sporozoites along with saliva are injected into a vertebrate host. The role of the host responses to mosquito saliva components in malaria remains unclear. We observed that antisera against Anopheles gambiae salivary glands partially protected mice from mosquito-borne Plasmodium infection. Specifically, antibodies to A. gambiae TRIO (AgTRIO), a mosquito salivary gland antigen, contributed to the protection. Mice administered AgTRIO antiserum showed lower Plasmodium liver burden and decreased parasitemia when exposed to infected mosquitoes. Active immunization with AgTRIO was also partially protective against Plasmodium berghei infection. A combination of AgTRIO antiserum and antibodies against Plasmodium circumsporozoite protein, a vaccine candidate, further decreased P. berghei infection. In humanized mice, AgTRIO antiserum afforded some protection against mosquito-transmitted Plasmodium falciparum. AgTRIO antiserum reduced the movement of sporozoites in the murine dermis. AgTRIO may serve as an arthropod-based target against Plasmodium to combat malaria.

Immunosurveillance of secondary lymphoid organs (SLO) is performed by central memory T cells that recirculate through blood. Resident memory T (Trm) cells remain parked in nonlymphoid tissues and often stably express CD69. We recently identified Trm cells within SLO, but the origin and phenotype of these cells remains unclear. Using parabiosis of "dirty" mice, we found that CD69 expression is insufficient to infer stable residence of SLO Trm cells. Restimulation of nonlymphoid memory CD8+ T cells within the skin or mucosa resulted in a substantial increase in bona fide Trm cells specifically within draining lymph nodes. SLO Trm cells derived from emigrants from nonlymphoid tissues and shared some transcriptional and phenotypic signatures associated with nonlymphoid Trm cells. These data indicate that nonlymphoid cells can give rise to SLO Trm cells and suggest vaccination strategies by which memory CD8+ T cell immunosurveillance can be regionalized to specific lymph nodes.

Chronic viral infections remain a global health concern. The early events that facilitate viral persistence have been linked to the activity of the immunoregulatory cytokine IL-10. However, the mechanisms by which IL-10 facilitates the establishment of chronic infection are not fully understood. Herein, we demonstrated that the antigen sensitivity of CD8+ T cells was decreased during chronic infection and that this was directly mediated by IL-10. Mechanistically, we showed that IL-10 induced the expression of Mgat5, a glycosyltransferase that enhances N-glycan branching on surface glycoproteins. Increased N-glycan branching on CD8+ T cells promoted the formation of a galectin 3-mediated membrane lattice, which restricted the interaction of key glycoproteins, ultimately increasing the antigenic threshold required for T cell activation. Our study identified a regulatory loop in which IL-10 directly restricts CD8+ T cell activation and function through modification of cell surface glycosylation allowing the establishment of chronic infection.

N-myc downstream-regulated gene 2 (NDRG2) is a differentiation- and stress-associated molecule that is predominantly expressed in astrocytes in the central nervous system. In this study, we examined the expression and role of NDRG2 in experimental autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis. Western blot and immunohistochemical analysis revealed that the expression of NDRG2 was observed in astrocytes of spinal cord, and was enhanced after EAE induction. A comparative analysis of wild-type and Ndrg2-/- mice revealed that deletion of Ndrg2 ameliorated the clinical symptoms of EAE. Although Ndrg2 deficiency only slightly affected the inflammatory response, based on the results of flow cytometry, qRT-PCR, and immunohistochemistry, it significantly reduced demyelination in the chronic phase, and, more importantly, neurodegeneration both in the acute and chronic phases. Further studies revealed that the expression of astrocytic glutamate transporters, including glutamate aspartate transporter (GLAST) and glutamate transporter 1, was more maintained in the Ndrg2-/- mice compared with wild-type mice after EAE induction. Consistent with these results, studies using cultured astrocytes revealed that Ndrg2 gene silencing increased the expression of GLAST, while NDRG2 over-expression decreased it without altering the expression of glial fibrillary acidic protein. The effect of NDRG2 on GLAST expression was associated with the activation of Akt, but not with the activation of nuclear factor-kappa B. These findings suggest that NDRG2 plays a key role in the pathology of EAE by modulating glutamate metabolism. Cover Image for this Issue: doi: 10.1111/jnc.14173.

Depression has been associated with a low-grade chronic inflammatory state, suggesting a potential therapeutic role for anti-inflammatory agents. Fisetin is a naturally occurring flavonoid in strawberries that has anti-inflammatory activities, but whether fisetin has antidepressant effects is unknown. In this study, we exposed mice to spatial restraint for 2 weeks with or without treatment with fisetin. Immobility time in the forced swimming and tail suspension test after this restraint increased in the untreated group, but this increase did not occur in the fisetin group. We administered fisetin to Abelson helper integration site-1 (Ahi1) knockout mice, which have depressive phenotypes. We found that fisetin attenuated the depressive phenotype of these Ahi1 knockout mice. We further investigated the potential mechanism of fisetin's antidepressant effects. Because TrkB is a critical signaling pathway in the mechanisms of depression, we examined whether phosphorylated TrkB was involved in the antidepressant effects of fisetin. We found that fisetin increased phosphorylated TrkB level without altering total TrkB; this increase was attenuated by K252a, a specific TrkB inhibitor. Taken together, our results demonstrated that fisetin may have therapeutic potential for treating depression and that this antidepressant effect may be mediated by the activation of the TrkB signaling pathway.

Protein kinase A (PKA) integrates inputs from G-protein-coupled neuromodulator receptors to modulate synaptic and cellular function. Gαs signaling stimulates PKA activity, whereas Gαi inhibits PKA activity. Gαq, on the other hand, signals through phospholipase C, and it remains unclear whether Gαq-coupled receptors signal to PKA in their native context. Here, using two independent optical reporters of PKA activity in acute mouse hippocampus slices, we show that endogenous Gαq-coupled muscarinic acetylcholine receptors activate PKA. Mechanistically, this effect is mediated by parallel signaling via either calcium or protein kinase C. Furthermore, multiple Gαq-coupled receptors modulate phosphorylation by PKA, a classical Gαs/Gαi effector. Thus, these results highlight PKA as a biochemical integrator of three major types of GPCRs and necessitate reconsideration of classic models used to predict neuronal signaling in response to the large family of Gαq-coupled receptors.

Post-stroke angiogenesis facilitates neurovascular remodeling process and promotes neurological recovery. Proangiogenic effects of Salvianolic acids (Sals) have been reported in various ischemic disorders. However, the underlying mechanisms are still poorly understood. Previous studies of our laboratory have demonstrated that activating Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway is involved in the protection against cerebral ischemia/reperfusion injury. In this study, we investigated the impacts of Sals on angiogenesis and long-term neurological recovery after ischemic stroke as well as the potential mechanisms. Male mice subjected to permanent distal middle cerebral artery occlusion were administrated with Sals, 5-bromo-2'-deoxyuridine, and JAK2 inhibitor AG490 once daily from day 1 to day 14 after distal middle cerebral artery occlusion. Compared with the control group, Sals treatment significantly improved neurological recovery at day 14 and 28 after ischemic stroke. Sals enhanced post-stroke angiogenesis, pericytes and astrocytic endfeet covered ratio in the peri-infarct area. The JAK2/STAT3 signaling pathway was activated by Sals in the angiogenesis process, and inhibition of JAK2/STAT3 signaling blocked the effects of Sals on post-stroke angiogenesis and neurological recovery as well as abolished the mediation of proangiogenic factors. In summary, these data suggest that Sals administration enhances cerebral angiogenesis and promotes neurological recovery after ischemic stroke, mediated by the activation of JAK2/STAT3 signaling pathway.

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.

Elucidating temporal windows of signaling activity required for synaptic and behavioral plasticity is crucial for understanding molecular mechanisms underlying these phenomena. Here, we developed photoactivatable autocamtide inhibitory peptide 2 (paAIP2), a genetically encoded, light-inducible inhibitor of CaMKII activity. The photoactivation of paAIP2 in neurons for 1-2 min during the induction of LTP and structural LTP (sLTP) of dendritic spines inhibited these forms of plasticity in hippocampal slices of rodents. However, photoactivation ∼1 min after the induction did not affect them, suggesting that the initial 1 min of CaMKII activation is sufficient for inducing LTP and sLTP. Furthermore, the photoactivation of paAIP2 expressed in amygdalar neurons of mice during an inhibitory avoidance task revealed that CaMKII activity during, but not after, training is required for the memory formation. Thus, we demonstrated that paAIP2 is useful to elucidate the temporal window of CaMKII activation required for synaptic plasticity and learning.

The potassium channel Kv1.3 is highly expressed in the mitochondria of various cancerous cells. Here we show that direct inhibition of Kv1.3 using two mitochondria-targeted inhibitors alters mitochondrial function and leads to reactive oxygen species (ROS)-mediated death of even chemoresistant cells independently of p53 status. These inhibitors killed 98% of ex vivo primary chronic B-lymphocytic leukemia tumor cells while sparing healthy B cells. In orthotopic mouse models of melanoma and pancreatic ductal adenocarcinoma, the compounds reduced tumor size by more than 90% and 60%, respectively, while sparing immune and cardiac functions. Our work provides direct evidence that specific pharmacological targeting of a mitochondrial potassium channel can lead to ROS-mediated selective apoptosis of cancer cells in vivo, without causing significant side effects.

Plasticity of adult neurogenesis supports adaptation to environmental changes. The identification of molecular mediators that signal these changes to neural progenitors in the niche has remained elusive. Here we report that diazepam binding inhibitor (DBI) is crucial in supporting an adaptive mechanism in response to changes in the environment. We provide evidence that DBI is expressed in stem cells in all neurogenic niches of the postnatal brain. Focusing on the hippocampal subgranular zone (SGZ) and employing multiple genetic manipulations in vivo, we demonstrate that DBI regulates the balance between preserving the stem cell pool and neurogenesis. Specifically, DBI dampens GABA activity in stem cells, thereby sustaining the proproliferative effect of physical exercise and enriched environment. Our data lend credence to the notion that the modulatory effect of DBI constitutes a general mechanism that regulates postnatal neurogenesis.

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